1. Field of the Invention
This invention relates to a device that quickly, accurately and reproducibly dilutes and mixes two or more solutions to provide incremental concentrations that can be analyzed by any appropriate analyzer.
In a preferred embodiment, this invention relates to a device which is capable of reproducibly mixing one reactant, such as an enzyme, with successively increasing concentrations of another (selected) reactant, so as to produce a series of reacting solutions varying only in the concentration of the selected reactant. The series of reacting solutions so produced can then be monitored to obtain kinetic data on the reaction. The invention further relates to an automated system comprising: the device connected to a stepping motor so as to rapidly produce a series of solutions varying only in the concentration of the selected reactant. The device can be optionally connected to a diode array spectrophotometer capable of simultaneously measuring the rate of reaction at one wavelength and the initial concentration of the selected reactant at another wavelength.
The device is useful because it provides a means for rapidly, accurately and reproducibly obtaining data on chemical, biochemical, and physical chemical reactions in solution.
2. Description of Related Art
The characterization of an enzyme by its Michaelis-Menten constant (Km), its turnover rate (V.sub.max) and its affinity constant (Ka) for activator and inhibitors is a labor intensive and tedious process. It typically requires the initial preparation of two or more master solutions. One of the master solutions, containing the selected reactant, is manually diluted down to produce a series of solutions varying in the concentration of the selected reactant. The appropriate solutions are then manually combined, mixed and introduced into a spectrophotometer to obtain a series of raw data from which the constants can be calculated. The preparation of the multiple dilutions, the manual combining and mixing of solutions, and the subsequent introduction of the combined solutions into a spectrophotometer is not only laborious but the number of manual steps involved makes the process subject to error as well.
It is the object of the present invention to provide a device capable of diluting a selected reactant and of combining and mixing the selected reactant with one or more reactants; said device being connected to a means for introducing the combined solution into an analyzer, such as a spectrophotometer, for monitoring and/or analysis. The overall invention thereby saving both time and labor.
Included among the prior art mixing devices are the gradient producing devices employed in high pressure liquid chromatography. Such devices produce a continually changing solution of eluent by mixing two solvents whereby the percent concentration of both solvents is slowly and gradually changed over time.
The present invention differs from the prior art gradient device in that the present invention is not directed toward gradually producing minute changes in the preparation of both solvents over time. Rather, the present invention is directed toward rapidly producing significant incremental changes in the concentration of a single selected reactant while keeping the concentrations of all other reactants constant.
The prior art mixing of an enzyme solution with its substrate includes devices which gently bubble a gas through a solution into which enzyme and/or substrate solutions have been added. An additional prior art mixing device teaches the mixing of a converged stream of flowing enzyme and substrate by causing the converged stream to flow in a long coil along a horizontal axis wherein segments of the flowing stream are separated from another by the insertion of a gas bubble. In this latter system, mixing is primarily accomplished by employing two solutions of different densities wherein gravity pulls the more dense solution towards the bottom of a flowing stream which is continuously inverted as it passes through the horizontally axised coils.
Unlike the prior art, the present invention accomplishes the mixing of combined and flowing streams of enzyme and substrate by repeatedly diverging and reconverging the flowing streams as they pass through a plurality of diverging and converging fluid passageways. In the present system, mixing occurs as a result of the turbulence created when the divergent streams are repeatedly converged at oblique angles.
The prior art also teaches stopped flow spectrometry, a technique wherein a drive system is used to mix equal volumes of reactants to produce a plurality of solutions of a single unvarying concentration which are propelled into a cell of a spectrophotometer. This prior art device differs from the present invention in that the prior art neither permits the continuous dilution of one of the reactants so as to provide a series of solutions varying only in the concentration of a single reactant nor provides a means for obtaining kinetic data enabling the characterization of an enzyme within a short period of time. Moreover, the prior art does not suggest that such dilutions are possible.